GENECLEAN Turbo for PCR Kit For purification of PCR products ranging from 100 bp to 10 kb

Transcription

1 GENECLEAN Turbo for PCR Kit For purification of PCR products ranging from 100 bp to 10 kb Revision # E09

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3 GENECLEAN Turbo for PCR Kit For purification of PCR products ranging from 100 bp to 10 kb Application Manual Revision # E09 Cat. Nos , 50 preps , 100 preps , 300 preps Any Questions? Call Technical Support at (800)

4 TABLE OF CONTENTS 1. Introduction Applications for GENECLEAN Turbo Technology How Does GENECLEAN Technology Work? Kit Components and User Supplied Materials GENECLEAN Turbo for PCR Kit Components User Supplied Materials Important Considerations Before Use Preparation of GENECLEAN Turbo for PCR Wash Binding Capacity Yield Measurements Simplified Protocol for Experienced Users Detailed Protocol Common Questions Does GENECLEAN Turbo for PCRwork on all conformations of plasmid DNA? Can I substitute GENECLEAN Turbo for PCR Wash Concentrate for other GENECLEAN Solutions? If I m using GENECLEAN Turbo for PCR, what do I do if I have more than 5 µg of DNA? Troubleshooting Low or No Recovery with the GENECLEAN Turbo for PCR Kit Problems with Binding Problems with Washing Problems with Eluting Rapid Kit Reagent Test Procedure Problems Measuring Yield visit us on the web at

5 7.2 Removing Primer Dimers (~50 bp) from Desired Fragment of 100 bp or More Replacing GENECLEAN Turbo for PCR Wash Solution References Recommended Reference Format Related Products Gel Isolation and Reaction Cleanup Products GENECLEAN -Based Genomic DNA Isolation Kits Plasmid Purification Products TRIPLE CHECK Tips Product Use Limitation & Warranty Any Questions? Call Technical Support at (800)

6 1. Introduction The GENECLEAN Turbo for PCR Kit is an advanced adaptation of the original GENECLEAN kit containing the patented GLASSMILK nucleic acid binding matrix. It uses a novel and patented GENECLEAN Turbo Cartridge system designed to further simplify the purification process. This system contains a special GLASSMILK -embedded membrane and buffer system optimized for the purification of PCR products from 100 bp to 10 kb. A carefully engineered hole in the Cartridge cap allows the addition of solutions without removing and closing the cap each time. A second hole in the outer portion of the cap permits the removal of waste solution from the Catch Tube using vacuum aspiration without removing the Turbo Cartridge from the Catch Tube. DNA is eluted in 30 µl of H 2 0 or TE buffer. Turbo Cartridges also have a luer lock fitting for use with any vacuum manifold. 1.1 Applications for GENECLEAN Turbo for PCR Technology Desalting Eliminate proteins from enzymatic reactions Remove primers and unincorporated nucleotides from enzymatic reactions Separate linearized from uncut vectors Isolate PCR product away from genomic DNA and primers Clean DNA before sequencing, transfection, transformation and microinjection 1.2 How Does GENECLEAN Technology Work? DNA generally binds to silica in high concentrations of chaotropic salt and elutes when the salt concentration is lowered. The mechanism of DNA binding to silica in high salt has not been completely described, but may involve chaotropic salt disruption of the water structure around negatively charged silica, allowing a cation bridge to form between it and the negatively charged phosphate backbone of DNA (see figure 1). When the salt concentration is lowered, rehydration of the silica matrix breaks the attraction between the matrix and DNA. The fact that DNA binds in high salt and elutes in low salt makes this method especially useful as a purification procedure. Since the DNA is eluted with either water or a low salt buffer, it can be used immediately in subsequent reactions without precipitation or other further manipulation. This is unlike ion exchange methods that require binding in low salt and elution in high salt and require precipitation or other means of removing salt before the DNA can be used. 6 visit us on the web at

7 Figure 1: Mechanism of DNA Binding to Silica 2. Kit Components and User Supplied Materials 2.1 GENECLEAN Turbo for PCR Kit Components GENECLEAN Turbo for PCR Salt Solution (28 ml, Cat. No ; 55 ml, Cat. No ; 165 ml, Cat. No ) is a specially prepared aqueous solution of a guanidine chaotropic binding salt that allows the DNA to bind to the GLASSMILK embedded in the GENECLEAN Turbo Cartridge. GENECLEAN Turbo for PCR Wash Concentrate (6 ml, Cat. No ; 11 ml, Cat. No ; 32 ml, Cat. No ) is a concentrated, proprietary salt solution to which ethanol is added to make GENECLEAN Turbo for PCR Wash (see Section 3.1). Store GENECLEAN Turbo for PCR Wash at the bench (15 30 C). Keep tightly capped to prevent evaporation of ethanol. GENECLEAN Turbo for PCR Cartridges (50, Cat. No ; 100, Cat. No ; 300, Cat. No ) contain a specially designed composite membrane that incorporates GLASSMILK as an integral part. The irregular shape of GLASSMILK, in addition to the thickness of the filter, provides a very large surface area and greater binding capacity than most silica-based filter media. Each column can bind up to 10 µg of DNA. The hole in the center of the cap allows for rapid delivery of solutions to the filter when the lid is closed and the hole in front allows for easy removal of liquid from the Catch Tube using vacuum aspiration. GENECLEAN Turbo for PCR Cartridges have a luer lock fitting so they can be used in a vacuum manifold (see Figure 2). Any Questions? Call Technical Support at (800)

8 Figure 2: Patented Turbo Cartridge System Two unique features of the GENECLEAN Turbo Cartridge for high throughput processing GENECLEAN Turbo Catch Tube Cartridge assembled unit Press a l ml pipet tip to #1 and add liquid in a steady stream. Aspirate liquid from catch tube through #2 without removing cartridge from catch tube. Use a blunted 2 inch long, 18 or less gauge, needle attached to a vacuum source such as a syringe or pump. # 2 # 1 It is best to practice these maneuvers before using them during the GENECLEAN Turbo for process. assembled unit (top view w/closed lid) GENECLEAN Turbo for PCR Catch Tubes (50, Cat. No ; 100, Cat. No ; 300, Cat. No ) are 1.5 ml microcentrifuge tubes with removable caps. They are used to recover and store DNA eluted from the GENECLEAN Turbo for PCR Cartridges. GENECLEAN Turbo for PCR Elution Solution (3.3 ml, Cat. No ; 6.6 ml, Cat. No ; 20 ml, Cat. No ) is RNase/DNase/pyrogen-free water for elution of DNA from the GENECLEAN Turbo for PCR Cartridges. 2.2 User Supplied Materials Benchtop microcentrifuge 200 proof (100%) ethanol Distilled water Vacuum aspirator (optional) Vacuum manifold (optional) 8 visit us on the web at

9 3. Important Considerations Before Use 3.1 Preparation of GENECLEAN Turbo for PCR Wash Before first use, add the correct volume of 200 proof (100%) ethanol to the GENECLEAN Turbo Wash Concentrate according to the chart below and mix well. Do not use denatured alcohol as it can cause precipitation of salts. Label the container and store tightly capped at C. Cat. No. No. of Preps Turbo Wash Concentrate 200 proof (100%) Ethanol ml 54 ml ml 99 ml ml 288 ml 3.2 Binding Capacity Each column can bind up to 10 µg of either ssdna or dsdna. 3.3 Yield Measurements The best method for checking yields of DNA isolated by GENECLEAN Turbo for PCR is to run an aliquot on an agarose gel using known quantities in adjacent lanes as controls. OD 260 and fluorescent readings can also be used to estimate yields, but these methods can be affected by trace amounts of salts and silica matrix. It is best to confirm these readings by gel analysis. 4. Simplified Protocol for Experienced Users 1. Transfer 100 µl oil-free PCR reaction to a 1.5 ml microcentrifuge tube. 2. Add 5 volumes GENECLEAN Turbo for PCR Salt Solution and mix. 3. Transfer PCR/Salt Solution to a GENECLEAN Turbo for PCR Cartridge. 4. Centrifuge for 5 seconds. Empty Catch Tube as needed. 5. Add 500 µl prepared GENECLEAN Turbo for PCR Wash to the filter. 6. Centrifuge for 5 seconds. Empty Catch Tube as needed. 7. Empty Catch Tube. Centrifuge the GENECLEAN Turbo for PCR Cartridge for an additional 4 minutes to remove residual Wash Solution. 8. Remove cap from Catch Tube, insert GENECLEAN Turbo for PCR Cartridge containing bound DNA. 9. Add 30 µl GENECLEAN Turbo for PCR Elution Solution directly onto GLASSMILK -embedded membrane and incubate at room temperature for 5 minutes. 10. Centrifuge for 30 seconds to transfer eluted DNA to GENECLEAN Turbo for PCR Catch Tube. Discard GENECLEAN Turbo for PCR Cartridge and cap the Catch Tube. Any Questions? Call Technical Support at (800)

10 5. Detailed Protocol 1. Transfer 100 µl oil-free PCR reaction to a 1.5 ml microcentrifuge tube. Spot the PCR sample onto parafilm and transfer the aqueous part 2-3 times to a new location on the parafilm, leaving the oil behind. At the last transfer, only the aqueous phase should be left. 2. Add 5 volumes GENECLEAN Turbo for PCR Salt Solution and mix by tapping side of tube with a finger. 3. Transfer PCR reaction/salt Solution mixture to a GENECLEAN Turbo for PCR Cartridge assembled in a kit-supplied 2 ml cap-less Catch Tube. 4. Centrifuge at <14,000 x g for 5 seconds. Empty Catch Tube as needed. Vacuum Manifold Option: Place the GENECLEAN Turbo for PCR Cartridge on a vacuum manifold. Apply vacuum to <25 inches Hg to completely drain liquid. 5. Add 500 µl of prepared GENECLEAN Turbo for PCR Wash to the filter.* [Note: Save time by bringing pipet tip in contact with opening #1 to deliver solution while the tube is in the microcentrifuge.] *VERY IMPORTANT: Be sure ethanol has been added to the GENECLEAN Turbo for PCR Wash Concentrate before using. See Section 3.1 for instructions. 6. Centrifuge at <14,000 x g for 5 seconds. Empty the Catch Tube as needed. [Note: Save time by aspirating liquid through opening #2 with a vacuum needle.] Vacuum Manifold Option: Place the GENECLEAN Turbo for PCR Cartridge on a vacuum manifold. Apply vacuum to <25 inches Hg until Cartridge is emptied of Wash. Repeat this step until all the Wash solution has passed through the filter. Optional: Repeat Wash procedure as detailed in Steps 5 & Empty the Catch Tube and centrifuge the GENECLEAN Turbo for PCR Cartridge at <14,000 x g for an additional 4 minutes to drive the last of the Wash Solution from the Turbo Cartridge. [Note: Centrifugation in an empty Catch Tube in this step is essential for removing trace alcohol from the final product.] 8. Remove the detachable cap from a GENECLEAN Turbo for PCR Catch Tube and set it aside. Insert the GENECLEAN Turbo for PCR Cartridge containing the bound DNA. 10 visit us on the web at

11 9. Add 30 µl GENECLEAN Turbo for PCR Elution Solution directly onto the GLASSMILK -embedded membrane and incubate for 5 minutes at room temperature. 10. Centrifuge at <14,000 x g for 30 seconds to transfer eluted DNA to GENECLEAN Turbo for PCR Catch Tube. Discard GENECLEAN Turbo for PCR Cartridge and cap the Catch Tube. DNA is now ready to use without further manipulation. [Note: A second elution is not necessary or recommended. Repetition of this step will cause the total volume to increase and the concentration of DNA to decrease.] 6. Common Questions 6.1 Does GENECLEAN Turbo for PCR work on all conformations of plasmid DNA? Our line of Plasmid Purification Kits would be best-suited for purification of plasmid DNA. See Section 10 for related products. 6.2 Can I substitute GENECLEAN Turbo for PCR Wash Concentrate for other GENECLEAN Solutions? No. The wash solutions have different salt concentrations and are prepared differently. 6.3 If I m using GENECLEAN Turbo for PCR, what do I do if I have more than 10 µg of DNA? Our line of slurry-based purification kits would be best-suited for larger quantities of DNA (specifically GENECLEAN, GENECLEAN II, or GENECLEAN III Kits.) See Section 10 for related products. 7. Troubleshooting 7.1 Low or No Recovery with the GENECLEAN Turbo for PCR Kit Problems with Binding Yields can be affected by insufficient incubation and mixing time during the DNA/Salt solution binding step. Inversion of the tube to mix the solution to homogeneity before loading the GENECLEAN Turbo for PCR Cartridge will increase efficiency Problems with Washing DNA may elute in the Wash if ethanol was not added to the GENECLEAN Turbo for PCR Wash Concentrate prior to use. Prepare GENECLEAN Turbo for PCR Wash as described in Section 3.1. If the GENECLEAN Turbo for PCR Wash ethanol concentration drops significantly due to evaporation, the DNA may elute in the Wash. Store the prepared GENECLEAN Turbo for PCR Wash tightly capped at C. Any Questions? Call Technical Support at (800)

12 7.1.3 Problems with Eluting Add GENECLEAN Turbo for PCR Elution Solution directly onto the GLASSMILK -embedded membrane and incubate 5 minutes at room temperature before recovering the DNA Rapid Kit Reagent Test Procedure If yields are less than 50%, this test takes minutes to determine if the problem is due to reagents or to some other aspect of the procedure. 1. Put µg of DNA into a final volume of 20 µl H 2 O or TE buffer. Transfer 10 µl into a microcentrifuge tube. 2. Add 50 µl GENECLEAN Turbo for PCR Salt Solution to the DNA in the microcentrifuge tube and mix well. 3. Transfer solution to a GENECLEAN Turbo for PCR Cartridge assembled into a 2 ml cap-less microcentrifuge tube. Centrifuge at <14,000 x g until all liquid has transferred to Catch Tube. Transfer the flowthrough to a new microcentrifuge tube. 4. Precipitate any DNA present in the flow-through by adding 50 µl water and 100 µl isopropanol and mix. Centrifuge at <14,000 x g for 5 minutes. Drain the tube and add 10 µl of water. 5. Add 300 µl GENECLEAN Turbo for PCR Wash and centrifuge. Save the Wash. 6. Centrifuge to dry for 2 minutes and transfer the GENECLEAN Turbo for PCR Cartridge to a new GENECLEAN Turbo for PCR Catch Tube. 7. Add 30 µl TE or H 2 O Solution directly onto the GLASSMILK -embedded membrane and incubate for 5 minutes at room temperature. Centrifuge at <14,000 x g for 1 minute and save supernatant. Transfer filter to a new Catch Tube. 8. Repeat elution (step 7). Save this supernatant to a new tube. 9. Run the following samples on a 0.8% agarose mini-gel until the samples migrate 1 2 cm. Lane 1: 5 µl DNA from the 10 µl left in step 1 that was not purified with the GENECLEAN Turbo for PCR. Lane 2: 15 µl first elution (step 7). Lane 3: 15 µl second elution (step 8). Lane 4: 10 µl GENECLEAN Turbo for PCR Wash (step 5, add Ficoll, sucrose, or glycerol to keep the GENECLEAN Turbo for PCR Wash in the well). Lane 5: 10 µl of the precipitated GENECLEAN Turbo for PCR supernatant after absorption (step 4). The results of the gel should show the fate of DNA during the GENECLEAN Turbo for PCR procedure. Most of the DNA should be in the first elution (lane 2), some (approximately 10%) should be in the second elution (lane 3), and none in GENECLEAN Turbo for PCR Wash or guanidine thiocyanate after absorption (lanes 4 and 5, respectively). If DNA is seen in lane 5, this indicates that not all the DNA bound to the Turbo Filter. DNA in lane 4 indicates loss during the Wash step. The relative quantities of DNA in each elution will indicate efficiencies during this step. The results of this rapid kit test normally show a recovery of 70% or more in the first elution. 12 visit us on the web at

13 7.1.5 Problems Measuring Yield DNA yield can be quantified with a fluorometer or estimated by running the sample against a known amount of DNA on an agarose gel. Using a spectrophotometer to quantify DNA yield is not recommended for the following reasons: 1. Residual silica particles (which do not interfere with downstream reactions or uses of the DNA) can scatter UV light, affecting OD 260 readings and OD 260 /OD 280 ratios. 2. After diluting part of your sample up to the minimum volume of the cuvette, the DNA will often be too dilute to give a significant reading. For example, if you eluted 0.5 µg in 20 µl of water and diluted 2 µl of this to 200 µl, the final concentration of DNA in the cuvette would be 0.5 µg/0.02 ml x 0.01=0.25 µg/ml. This would give an absorbance of only 0.005, which is too low to be significant on most instruments. 7.2 Removing Primer Dimers (~50 bp) from Desired Fragment of 100 bp or More To prevent the binding of primer dimers approximately 50bp in size, use diluted GENECLEAN Turbo for PCR Wash Solution. To prepare diluted wash add 220 µl H 2 O per 780 µl prepared GENECLEAN Turbo for PCR Wash Solution (see Section 3.1). Diluted wash should be used only when necessary as it may reduce recovery of the desired fragment. 7.3 Replacing GENECLEAN Turbo for PCR Wash Solution The amount of GENECLEAN Turbo for PCR Wash Concentrate provided is sufficient when the protocol is followed as recommended. If the protocol is changed such that more prepared GENECLEAN Turbo for PCR Wash Solution is required, a solution of 80% ethanol can be made using TE (10 mm Tris and 1 mm EDTA, ph 7.5) and will work nearly as well as the proprietary GENECLEAN Turbo Wash Solution. 8. References Many of the principles of the GENECLEAN procedures described here are based on the data of Vogelstein and Gillespie (1). See reference 2 for discussion of the effects of chaotropic salts and temperature on DNA stability. 1. Vogelstein, B. and Gillespie, D. (1979) Proc. Nat. Acad.Sci., USA. 76, Hamaguchi, K. and Geiduschek, E.P. (1962) J. Amer. Chem. Soc. 84, Recommended Reference Format DNA of kb was purified from solution using the GENECLEAN Turbo for PCR Kit (Qbiogene, Inc., Carlsbad, California). Any Questions? Call Technical Support at (800)

14 10. Related Products 10.1 Gel Isolation and Reaction Cleanup Products Cat. No. Description Size GENECLEAN Turbo Kit 50 preps GENECLEAN Turbo Kit 100 preps GENECLEAN Turbo for PCR Kit 50 preps GENECLEAN Turbo for PCR Kit 100 preps GENECLEAN Kit 200 preps GENECLEAN II Kit 300 preps GENECLEAN III Kit 600 preps GENECLEAN SPIN Kit 50 preps GENECLEAN SPIN Kit 100 preps GENECLEAN Turbo 96 Kit 96 preps GENECLEAN Turbo 96 Kit 384 preps MERmaid Kit 200 preps MERmaid SPIN Kit 25 preps MERmaid SPIN Kit 75 preps MERmaid SPIN Kit 150 preps RNaid Kit 200 preps RNaid SPIN Kit 200 preps SeqDirect PCR Cleaning Kit 16 reactions SeqDirect PCR Cleaning Kit 32 reactions SeqDirect 96 PCR Cleaning Kit 1 x 96-well plate EtBr GREENBAG Disposal Kit 50 bags xTAE GENECLEAN Grade Electrophoresis Buffer 1.0 L TBE GENECLEAN Grade Electrophoresis Buffer Mix 425 g TBE GENECLEAN Grade Electrophoresis Buffer Mix 1,700 g SPIN Module (Includes # ) 60 F/T SPIN Module (Includes # ) 100 F/T Label Block 1 ml 10.2 GENECLEAN -Based Genomic DNA Isolation Kits Cat. No. Description Size FastDNA Kit 100 preps FastDNA Kit for Soi 50 preps GNOME DNA Isolation Kit 25 preps GNOME DNA Isolation Kit 100 preps GNOME Whole Blood DNA Isolation Kit 100 preps 14 visit us on the web at

15 FLORACLEAN Kit 25 preps GENECLEAN for Ancient DNA Kit 100 preps Whole Cell Yeast PCR Kit 200 preps Yeast Cell Lysis Kit 100 preps λquick! Kit 25 preps ssphage DNA SPIN Kit 60 preps 10.3 Plasmid Purification Products Cat. No. Description Size RapidPURE Plasmid Mini Kit 60 preps RapidPURE Plasmid Mini Kit 120 preps RapidPURE Plasmid Mini Kit 300 preps RapidPURE Plasmid Mini 96 Kit 96 preps RapidPURE Plasmid Mini 96 Kit 192 preps RPM Kit 60 preps RPM Kit 120 preps RPM Kit 300 preps Yeast RPM Kit 100 preps MiniPrep Express Matrix 1,250 preps well Prep Express 384 preps RapidPURE Plasmid Midi Kit 25 preps RapidPURE Plasmid Midi Kit 75 preps RapidPURE Plasmid Midi Kit 150 preps RapidPURE Plasmid Maxi GF Kit 20 preps RapidPURE Plasmid Maxi GF Reagent Kit 20 preps RapidPURE Plasmid Maxi GF Endo Free Kit 10 preps RapidPURE Plasmid Giga Kit 12 preps 10.4 TRIPLE CHECK Tips Cat. No. Description Size TRIPLE CHECK Tips (non-sterile) Bag of TRIPLE CHECK Tips (non-sterile) Bag of 1, TRIPLE CHECK Tips (non-sterile) Bag of 5, TRIPLE CHECK Tips (non-sterile) Box of TRIPLE CHECK Tips (non-sterile) Box of TRIPLE CHECK Tips (non-sterile) 4 racks of TRIPLE CHECK Tips (non-sterile) 10 racks of TRIPLE CHECK Tips (sterile) 4 racks of TRIPLE CHECK Tips (sterile) 10 racks of 96 Any Questions? Call Technical Support at (800)

16 11. Product Use Limitation & Warranty Unless otherwise indicated, this product is for research use only. Purchase of Qbiogene, Inc. products does not grant rights to reproduce, modify, or repackage the products or any derivative thereof to third parties. Qbiogene, Inc. makes no warranty of any kind, expressed or implied, including merchantability or fitness for any particular purpose, except that the products sold will meet our specifications at the time of delivery. Buyer s exclusive remedy and the sole liability of Qbiogene, Inc. hereunder shall be limited to, at our discretion, no replacement or compensation, product credits, refund of the purchase price of, or the replacement of materials that do not meet our specification. By acceptance of the product, Buyer indemnifies and holds Qbiogene, Inc. harmless against, and assumes all liability for, the consequence of its use or misuse by the Buyer, its employees or others, including, but not limited to, the cost of handling. Said refund or replacement is conditioned on Buyer notifying Qbiogene, Inc. within thirty (30) days of receipt of product. Failure of Buyer to give said notice within thirty (30) days shall constitute a waiver by the Buyer of all claims hereunder with respect to said material(s). GENECLEAN, GLASSMILK, MERmaid, RNaid, RPM, TRIPLE CHECK, FastDNA, GNOME, FLOR- ACLEAN, λquick! and BIO 101 Systems are registered trademarks of Qbiogene, Inc. BioFloat, GREEN- BAG, MiniPrep Express, RapidPURE and SeqDirect are trademarks of Qbiogene, Inc. GENECLEAN products are covered under US patent # 6,027, visit us on the web at

17 NOTES Any Questions? Call Technical Support at (800)

18 NOTES 18 visit us on the web at

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